Drug metabolism problems such as production of toxic metabolites and unfavorable pharmacokinetics cause almost half of all drug candidate failures during clinical trials. Although glucuronidation is one of the most important routes of biotransformation, the broad and overlapping substrate specificity of the hepatic UDP-glucuronosyltransferases (UGTs) that catalyze glucuronidation remains poorly understood. The two main reasons for this situation are the lack of isolated individual UGT isozymes and the lack of assay methods suitable for detecting glucuronidation of diverse chemicals.
The UDP-glucuronosyltransferases are a family of enzymes that catalyze the glucuronidation of endogenous and xenobiotic chemicals (Equation 1), generating products that are more hydrophilic and thus more readily excreted in bile or urine.
Equation 1
Uridine diphosphate-glucuronic acid (UDPGA)+aglycone.fwdarw.UDP+glucuronide
The UGTs play a key role in several important metabolic functions, including:
elimination of drugs such as non-steroidal anti-inflammatories, opioids, antihistamines, antipsychotics and antidepressants, PA1 detoxification of environmental contaminants such as benzo(a)pyrenes, PA1 regulation of hormone levels for androgens, estrogens, progestins, and retinoids, PA1 elimination of the heme degradation product bilirubin. PA1 After isozyme identification, more detailed kinetic studies with the appropriate UGT isozyme can be used to predict in vivo clearance rates, reducing the number of compounds that fail in clinical studies due to poor pharmacokinetics. PA1 Knowledge of metabolism by a specific UGT alerts the drug discovery team to potential pharmacogenetic problems, since genetic differences in UGT levels are recognized as an important factor in varying responses to therapeutics. PA1 Identification of the UGT responsible for the metabolism of a drug will aid in judicious selection of the in vitro assays or animal models used for preclinical assessment of possible drug--drug interactions and toxicology testing, thereby reducing inappropriate or unnecessary use of animals for experiments. PA1 Metabolism data can be used as a component of rational drug design. A better understanding of the structure-activity relationships that define substrate specificity for the various UGT isozymes would provide a basis for structural modifications of primary compounds to change their metabolism profile. This approach was used successfully for development of ABT-761, a 5-lipoxygenase inhibitor. PA1 The testing of glucuronidated compounds can lead to the discovery of valuable prodrugs that are inactive until metabolized in the body into an active form.
Although glucuronidation generally is classified as Phase II metabolism--the phase occurring after P450 dependent oxidative metabolism--many compounds do not require prior oxidation because they already possess functional groups that can be glucuronidated. Examples of first pass metabolism catalyzed by UGTs include the UGT2B7-dependent glucuronidation of morphine and the glucuronidation of 5-lipoxygenase inhibitors (antiinflammatories); in the latter case glucuronidation was demonstrated to be the rate limiting step for in vivo plasma clearance.
Notably, glucuronidation does not always cause decreased biological activity and/or detoxification. Glucuronides of some drugs are toxic, and have been linked with adverse drug reactions including immune hypersensitivity. Glucuronidation can modulate the potency of some drugs: the 6-glucuronide of morphine is a more potent analgesic than the parent compound, whereas the 3-glucuronide is a morphine antagonist. In addition, steroid glucuronidation can produce more active or toxic metabolites under pathophysiological conditions or during steroid therapies.
UGTs are 50-60 kDa integral membrane proteins with the major portion of the protein, including the catalytic domain, located in the lumen of the endoplasmic reticulum and a C-terminal anchoring region spanning the ER membrane. Two UGT families--UGT1 and UGT2--have been identified in humans; although the members of these families are less than 50% identical in primary amino acid sequence, they exhibit significant overlap in substrate specificity.
The members of the UGT1 family that are expressed in human liver, where the majority of xenobiotic metabolism takes place, include UGT 1A1, 1A3, 1A4, 1A6, and 1A9. Although the UGT2 family has not been studied as extensively, it is known that UGT2B4, 2B7, 2B10, 2B15 and 2B17 are expressed in the liver. Mutations in UGTs are known to have deleterious effects, including hyperbilirubinaemia which occurs with a frequency of 5-12% and can lead to neurotoxicity and in severe cases, death. As is the case for other drug metabolizing enzymes such as P450s, interindividual differences in UGT expression levels have been observed and linked to differences in drug responses (17,18). For instance, low expression of UGT1A1, as in patients with Gilbert's syndrome, has been associated with the toxicity of Irinotecan, a promising anticancer agent. In addition, UGT upregulation in tumor tissues has been identified as a possible cause of anticancer drug resistance (20,21).
Specificity for Aglycones--UGT substrates are known as aglycones; the products of the reaction are called glucuronides. All of the known UGTs exhibit broad substrate specificity, with a single isozyme catalyzing glucuronidation of a broad range of structurally unrelated compounds; not surprisingly there also is a great deal of overlap in the specificities of UGT isozymes. The sites of glucuronidation generally are nucleophilic nitrogen, sulfur or oxygen atoms in functional groups such as aliphatic alcohols, phenols, carboxylic acids, primary through tertiary amines, and free sulfyhydryls. The aglycone binding site is believed to be in the N-terminal portion of the UGT polypeptide, the region of the protein that shows the greatest variability in sequence among isozymes. However, efforts to define the aglycone binding site by correlating N-terminal amino acid sequences of UGT isozymes with their substrate specificities have been unsuccessful.
Despite their broad substrate specificities, UGTs can be highly regio- and stereo-selective. It has been suggested that substrates bind loosely to a very "open" substrate binding pocket--as with some P450s--and rotate until reactive functional groups are suitably oriented to the bound UDPGA and the amino acids involved in catalysis. Although several studies on the substrate specificities of individual recombinant UGTs have been performed, most have been limited to a relatively small number of compounds within one or two structural classes.
HTS assay methods described herein can be used to rapidly screen large numbers of diverse chemicals thus allowing a systematic effort to fully define the "chemical space" recognized by each of the key hepatic UGTs. Moreover, these HTS assay methods will fulfill the immediate needs of the pharmaceutical industry by providing a means to screen large numbers of diverse compounds for glucuronidation with a panel of the key human UGT isozymes. The information obtained with these HTS assays can be used in the following ways:
To confirm the need for improved technology to probe the specificity of UGT isozymes, it is useful to review the methods currently employed for in vitro drug metabolism studies, and the reasons why they are not adequate for immediate drug discovery needs.
Sources of UGTs. The important drug metabolizing UGT isozymes are located in the endoplasmic reticulum of liver cells. Natural sources of UGT for in vitro assays include liver slices, cultured cells, and cell fractions such as human liver microsomes. The major drawbacks of these unpurified systems are that they contain a mixture of multiple UGT isozymes and other drug metabolizing enzymes. As a result, they are of limited use in obtaining meaningful data on a specific UGT isozyme--particularly in an HTS format. Heterologous expression systems such as mammalian and BaV-infected insect cells have made it possible to produce large amounts of microsomal membranes highly enriched in a single UGT isozyme.
Assay methods. UGTs generally are assayed by isolation and quantification of the radioactively labeled metabolites produced from the parent compound in reactions containing radiolabeled UDPGA. In most cases, this involves chromatographic techniques such as thin layer chromatography (TLC) or high pressure liquid chromatography (HPLC), and in some cases phase separations. There are two major drawbacks to these assays methods. First, the need to isolate the reaction products makes the methods too cumbersome and time consuming for use in any type of high volume assay format. Second, different glucuronidated metabolites have different chromatographic properties, raising an obvious technical barrier to screening diverse compounds for metabolism by a panel of isolated UGT isozymes. For some substrates, products and reactants can be differentiated on the basis of altered absorbance or fluorescence after glucuronidation, however, these methods are limited to a few UGT isozymes.